Cervical carcinoma is considered to be the third most common cancer in women in the world. In 1994 an estimated 55.000 women in the U.S. were diagnosed with carcinoma in situ of the cervix, with an additional 15.000 cases of invasive cancer. Although organized or voluntary screening is available in a number of countries and a range of interventions exist, about 4.600 of women diagnosed with the disease do not survive. In Sweden organized screening has been in operation for the last 20 years, but still about 500 cases of invasive cancer are diagnosed annually. Although in the US and Europe major progress has been made in the control of cervical cancer, it remains a significant cause of morbidity and mortality in the developing world.
Infection by certain types of human papillomavirus (HPV) is the single most important risk factor for the development of cervical cancer. More than 95% of cervical cancer biopsies have been found to contain DNA of high-risk HPV types, most commonly HPV 16, followed by HPV 18, 45, 31 and 33. Given the importance of HPV infection in the etiology of cervical cancer, a large number of methods have been developed for detecting of the virus or for identifying the cellular changes resulting from viral transformation. Serological detection methods have been used to detect present or recent infection with HPV, but have a limitation in that not every infected individual develop antibodies. A number of DNA technologies have been employed for detection of viral nucleic acid, such as in situ hybridization, restriction fragment length polymorphism (RFLP) and southern-blot analysis, hybrid capture (where a DNA-RNA heteroduplex is recognized by monoclonal antibodies) and various PCR based assays. Many of the PCR systems developed for HPV detection involve an amplification step followed by a separate step for identification of individual HPV types To increase the technical sensitivity of the assay when analyzing samples with limited DNA, such as formalin-fixed biopsies or archival Papanicolaou (Pap) cervical smears, a nested-PCR has frequently been employed.
Previously an assay based on real-time PCR for the detection and quantification of high risk HPV DNA has been described (Josefsson et al., 1999). The 5′ exonuclease assay, employed in real-time PCR, is based on the ability of the 5′ to 3′ exonuclease activity of Taq polymerase to cleave a dual-labeled, non-extendible, hybridization probe during the extension phase of the PCR.
Using this previously described method it was demonstrated, in a case-control study, that the titer of HPV 16 in cervical smears can be used to predict the risk of development of cervical cancer in situ (cervical interstitial neoplasia, stage II; CIN III) (Josefsson et al., 2000; Ylitalo et al., 2000). These results indicate that HPV titer may represent a powerful means of determining whether an infection will progress into cervical cancer or be cleared. This invention is described in U.S. Pat. No. 6,420,106 and relates to a method to predict the risk of progression to virus associated cancer in a human subject.